Agradecemos al Dr. William Cashdollar, y la Fundación Centro de Imagen Northwestern Mutual en el Instituto de Investigación de la Sangre de Wisconsin para ayudar con los estudios de imagen. También agradecemos al Dr. Daria Ilatovskaya para la lectura crítica de este manuscrito y útil para el debate. Este estudio fue apoyado por los Institutos Nacionales de Salud Subvenciones HL108880 (a AS) y DP2-OD008396 (a AMG).

Los procedimientos experimentales se describen a continuación fueron aprobados por el Cuidado y Uso de Animales Comité Institucional (IACUC) en el Colegio Médico de Wisconsin y estaban de acuerdo con los Institutos Nacionales de Salud de Guía para el Cuidado y Uso de Animales de Laboratorio. 1. Aislamiento de la aorta de rata <ol><li> Anestesiar las ratas con isoflurano (5% de inducción, 1,5 a 2,5% de mantenimiento) u otro método aprobado IACUC. </li><li> Coloque la rata en una posición supina. Con el fin de exponer los órganos abdominales, hacer una pequeña incisión en la línea media ventral a través de la piel y del tejido subcutáneo abdominal. Uso de almohadillas de gasa desvían suavemente los intestinos para exponer la aorta y la vena cava como se muestra en la Figura 1. </li><li> Brevemente, embotado diseccionar la aorta desde el tejido conectivo y de la vena cava. El uso de sutura, atar la aorta lo más cerca posible al riñón. Diseccionar unos 1-2 cm de aorta y colocarlo en una cónica 15 ml maser contiene fisiológico normal de Salt Solution (PSS; en mM: 140 NaCl, 1,2 de MgCl 2, 2 CaCl 2, 4,5 KCl, 6 glucosa, 10 HEPES) en hielo. </li><li> Una vez que la aorta se aísla, la eutanasia del animal de acuerdo con los protocolos del IACUC aprobados. A la finalización de todos los no-supervivencia procedimientos eutanasia animales profundamente anestesiados por toracotomía inducir neumotórax para asegurar la desaparición humano del animal. 3 </li><li> Fórceps Utilizando un microscopio de disección, con punta fina, y microtijeras diseccionar la grasa y el tejido conectivo fuera de la aorta. Una vez que la aorta es limpio, cortar la aorta longitudinalmente y colocarlo en PSS en hielo para después. </li></ol> 2. colorante de carga e Incubación <ol><li> Pipet 7 l de 1 mM Fluo-04 a.m. tinte, que se suministra en la solución basada en DMSO, en 500 tubos individuales mu l que se han cubierto con papel de aluminio. Almacenar en el congelador a -20 ° C para preservar las propiedades de tinte con el tiempo. Nota: estas alícuotas de modoluciones deben ser estables durante al menos seis meses. </li><li> Antes de usar, permiten que las soluciones de colorante para calentar a temperatura ambiente antes de abrir. Preparar soluciones de trabajo inmediatamente antes de su uso. No almacene el reactivo diluido para su uso posterior. </li><li> Añadir 7 l de mM Fluo-04 a.m. tinte 1 confeccionada disuelto en solución de DMSO al 450 ul de PSS que no contiene calcio. Añadir 40 l de 10% de solución de DMSO no a base de ácido Pluronic al cóctel de carga para ayudar a dispersar los acetoximetilo (AM) ésteres y mejorar la carga del colorante de calcio. </li><li> Colocar las aortas disecados en los tubos 500 l que contenía el cóctel de carga (como se describe en 2.3) durante 1 h. Cubra todos los tubos con papel y lugar en un agitador rotatorio a temperatura ambiente. </li><li> Para supervisar la producción de NO en la vasculatura, utilizar colorante diacetato DAF-FM. Incubar la aorta en una solución que contiene 450 l PSS, 40 l de ácido plurónico y 5 l de 10 mM diacetato de DAF-FM. Similar al protocolo de incubación de calcio (descrito en 2.3), colocar la aortaen un tubo de 500 l que contiene la solución NO-dye cubierta en papel de aluminio y colocar en un agitador rotatorio durante 30 min a 4 ° C, seguido de la próxima 30 min a temperatura ambiente. </li><li> Cuando se mide la producción de NO, utilizar el NO endotelial bloqueador sintasa, L-NAME, para bloquear la producción de NO como un control como se muestra en la Figura 4B. Para este procedimiento, se suman a cóctel (como se describe en 2.5) 100 nM L-NAME durante los últimos 30 minutos de incubación (a temperatura ambiente). </li></ol> 3. Protocolo de fotones Microscopía de escaneo láser Dos <ol><li> Después de la 1 h de incubación, lavar la aorta 2 - 3 veces en PSS limpia para eliminar tinte extracelular. </li><li> Transferir la aorta a un plato recubierto de silicona que contiene ya sea normal o PSS Ca 2+ tampón libre (depende del protocolo experimental). Pin de la aorta hacia abajo sobre el revestimiento de silicona con la adventicia en contacto con la silicona (es decir, lumen endoteliales expuestas a la abertura de plato) utilizando pasadores en forma de μ. También puede utilizar un ancla de la rebanadarejilla o pegamento para fijar el tejido. Nota: Para reducir el estrés y posibles artefactos mecánicos, traslado y fijar aorta en Ca 2 + libre de solución y esperar 5-10 minutos antes de comenzar el experimento. </li><li> Conectar una bomba peristáltica o una jeringa a la placa recubierta de silicona para la aplicación automática o manual de fármacos o para cambiar la solución extracelular. </li><li> Coloque el plato debajo de la pieza de la nariz de los rectos microscopio de dos fotones, e instalar y posicionar un 25 × (NA 1.05 y el trabajo a distancia 2 mm) de inmersión en agua lente objetivo por encima de la muestra. Nota: Si este objetivo específico no está disponible, utilice un objetivo fabricados específicamente para dos imágenes de fotones, ya que puede aumentar la resolución de la señal de forma espectacular en comparación con un objetivo normal. </li><li> Activar el láser de dos fotones usando el software (el láser comienza a bombear y encienda). Sintonice la excitación láser de 820 nm. </li><li> El uso de epifluorescencia, busque la aorta y se centran en el objetivola superficie endotelial utilizando ajuste grueso. </li><li> Cambie el microscopio en el modo de escaneo láser de dos fotones, lo que garantiza que el láser de excitación es el modo de bloqueo, los detectores no descanned están comprometidos y se instalan los filtros de emisión correspondientes a los espectros de emisión esperada. Nota: Aquí, la FV10-MRL / R (495-540 nm) se utilizaron. </li><li> Iniciar escaneo en vivo utilizando aproximadamente 5% de la potencia del láser y finamente enfocar el objetivo con el fin de visualizar las células endoteliales. Nota: Las células endoteliales exhibirán una fuerte señal fluorescente cuando se incubaron en PSS normal. Este será notablemente más débil cuando los vasos se incubaron en tampón libre de calcio. Las células endoteliales estarán presentes en la parte superior de la aorta se abrió y las células musculares lisas se pueden ver justo debajo de las células endoteliales (ver Figura 2). Debido a que los vasos sanguíneos no son ni lisa ni incluso, habrá lugares en los que tanto las células de músculo liso y células endoteliales están presentes. &lt;/ Li&gt; <li> Una vez que las células están en foco, el programa de software microscopio para recoger imágenes secuenciales en el modo de exploración rápida (exploración de trama, ventana 512 x 512 píxeles con una frecuencia marco de la colección de 1,1 s). En paralelo con Fluo-4 a.m. fluorescencia, recopilar imágenes de luz transmitida con el fin de detectar cambios en la contracción de los vasos y visualizar mejor las condiciones experimentales. </li><li> Después de la recogida de imágenes de señal de referencia, cambiar la concentración de iones Ca2 + en la solución externa o lentamente aplicar agentes farmacológicos de estímulo usando la bomba peristáltica mientras que las imágenes. Observe el aumento de la señal fluorescente dentro de las células cargadas. Nota: Las células endoteliales responden a cambios en el flujo, lo que se recomienda utilizar aplicaciones laminares bajos y de prueba del vehículo antes de la aplicación de los activadores farmacológicos de Ca 2+ o NO señalización. Para volver a calcular el calcio intracelular a los valores de concentración nM en vasijas cargadas con Fluo-4 (constante de disociación de Fluo-4 is 345 nM), la intensidad de fluorescencia puede ser registrada al inicio del estudio y después de la adición de ionomicina y MnCl2. 4 </li></ol> 4. Procesamiento de Imágenes y Cálculos <ol><li> Descargue e instale Fiji paquete de procesamiento de imagen. </li><li> Abra el archivo de imagen en Fiji. Al importar el archivo, dividir los canales (luz transmitida y la señal fluorescente) cuando se le solicite. Utilice únicamente la señal fluorescente para el análisis de datos. </li><li> Dentro del programa de Fiji, haga clic en la ficha analizar y desplácese hacia abajo hasta la opción herramientas. Bajo la opción de herramientas, encontrará la ficha que dice el gerente de retorno de la inversión y haga clic en él; Aparecerá una nueva ventana. </li><li> Después de esto, haga clic en las mediciones establecidas en la ficha analizar. Seleccione las mediciones específicas de interés. Para las medidas transitorias de calcio, utilice la opción del valor medio gris. </li><li> Con la herramienta de trazo circular, comenzar a trazar las regiones de interés (ROI) y haga clic en &quot;añadir&quot; en la pantalla del gestor de retorno de la inversión para cada ROI. Una vez que todas las células de interés se han trazado, en la ventana del gestor de ROI, seleccione Medir Multi bajo la etiqueta de &quot;más&quot;. Recuerde que debe guardar bien las mediciones directamente o copiar y pegar todas estas mediciones en una hoja de cálculo. </li><li> Abrir archivo * .txt o Excel hoja de cálculo desde Fiji y transferir los números de una nueva hoja de Origen. Nota: También puede utilizar cualquier programa como Sigma Plot o Prisma para su posterior análisis. </li><li> Dentro del programa de origen, utilice Parcela → Menú Line + Símbolo de observar una visión general de todas las respuestas transitorias. Anule la selección de las células que no responden. </li><li> Volver a calcular el número de seguimiento para escala de tiempo (eje X), utilice la columna → Establecer valores de columna y multiplicar la columna el número de seguimiento para la frecuencia de recogida de imagen (en nuestro caso 1.1s). </li><li> Utilice Análisis → Estadísticas en filas para todas las grabaciones seleccionadas para calcular media (eje Y) y trazas de error estándar. Trazar el gráfico final para el grupo actual de células Parcela → Línea + Símbolo. </li><li> El mean calcio cálculo transición se describe como sigue. <ol><li> Para liberación total de calcio dentro del programa de origen, haga clic en el gráfico, a continuación, utilice el menú Análisis → Cálculo → Integrar. Integral total del área bajo la curva aparece en registro de resultados. </li><li> Para la cinética transitoria, haga clic en el gráfico, a continuación, utilizar el menú: Curva Análisis → no lineal Fit → Seleccionar Conjunto de Datos (elegir eje X van desde el máximo hasta el final de la respuesta transitoria). Iniciar montaje → Seleccionar Función → Exponencial Decay 1 → Hecho. Nota: ajuste exponencial aparece en la ventana de gráficos y registro de resultados. Los datos obtenidos (valores) representan cantidad total de calcio liberado, y la cinética de los canales mediadas la respuesta transitoria. </li></ol></li></ol>

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Descripción general Experimental. Para comprender mejor la contribución de calcio y NO a la fisiología vascular, un nuevo método fue desarrollado para la medición de [Ca2 +] i y NO en el músculo liso y las células endoteliales de aortas intactas aisladas. Juntos, este protocolo consta de estos pasos críticos: 1) aislamiento mecánico y preparación (digestión no enzimática) de la embarcación. Es importante mantener el tejido sano y intacta tanto como sea posible para obtener gra...

El calcio es un segundo mensajero fundamental dentro de las células vasculares tales como células endoteliales y musculares lisas. Es el estímulo primario para la contracción vascular y desempeña un papel importante en la dilatación vascular, incluyendo sus efectos a través de la generación de NO en el endotelio. Debido a las limitaciones de las tecnologías de formación de imágenes, ha sido prácticamente imposible observar el manejo del calcio dentro del recipiente intacto. El desarrollo de dos sistemas de imagen de fotones y la creación de nuevos calcio o NO tintes de etiquetado, hace posible la imagen a la profundidad y la resolución suficiente para empezar a entender la dinámica del calcio y la producción de NO dentro de la vasculatura. Microscopía de dos fotones se ha aplicado recientemente en los tejidos, los órganos y los estudios en animales enteros, incluso debido a su capacidad superior para penetrar profundamente los tejidos con baja fluorescencia de fondo y la alta sensibilidad de la señal. 1,2 El estrecho espectro de excitación de dos fotones en el Iluminadorpunto focal de iones y el uso de detectores no descanned son las razones por las dos microscopía fotón es superior a la microscopía confocal tradicional. La microscopía confocal no puede producir imágenes de alta calidad en la profundidad del tejido necesario debido a la auto-fluorescencia y la dispersión de la luz fuera de foco en el agujero confocal. En consecuencia, hemos desarrollado un método que utiliza un microscopio de dos fotones para medir [Ca 2 +] i la señalización y la producción de NO en células intactas, los vasos sanguíneos individuales con una baja relación señal-ruido de alta resolución y.

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	<tbody>
		<tr>
			<td>DAF-FM</td>
			<td>Life Technologies</td>
			<td>D-23842</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo 4 AM</td>
			<td>Life Technologies</td>
			<td>F14217</td>
			<td>500&#181;l in DMSO</td>
		</tr>
		<tr>
			<td>Pluronic F-68 solution</td>
			<td>Sigma-Aldrich</td>
			<td>P5556</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Name</td>
			<td>Tocris</td>
			<td>0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus upright microscope</td>
			<td>Olympus</td>
			<td>Fluoview FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MaiTai HP DeepSee-OL&#160;</td>
			<td>Spectra Physics</td>
			<td></td>
			<td>Ti:sapphire laser 690nm &#8212; 1040nm</td>
		</tr>
		<tr>
			<td>Filters</td>
			<td>Olympus</td>
			<td>FV10-MRL/R&#160;</td>
			<td>495 to 540 nm&#160;</td>
		</tr>
		<tr>
			<td>25&#215; water-immersion objective lens</td>
			<td>Olympus</td>
			<td>XLPL25XWMP</td>
			<td>N.A. 1.05 and working distance 2 mm</td>
		</tr>
		<tr>
			<td>Slice Anchor grid&#160;</td>
			<td>Warner Instrument&#160;</td>
			<td>SHD-27LH</td>
			<td></td>
		</tr>
		<tr>
			<td>Other basic reagents&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

two-photon imaging of intracellular ca2+ handling and nitric oxide production in endothelial and smooth muscle cells of an isolated rat aorta

El calcio es un regulador muy importante de muchos procesos fisiológicos en los tejidos vasculares. La mayoría de las funciones endoteliales y de músculo liso dependen en gran medida de los cambios en el calcio intracelular ([Ca2 +] i) y óxido nítrico (NO). Con el fin de entender cómo [Ca 2 +] i, NO y moléculas de aguas abajo son manejados por un vaso sanguíneo en respuesta a vasoconstrictores y vasodilatadores, hemos desarrollado una nueva técnica que se aplica en calcio etiquetado (o NO-etiquetado) tiñe con dos fotones microscopía para medir el manejo del calcio (o la producción de NO) en los vasos sanguíneos aislados. Descrito aquí es un procedimiento que se detalla paso a paso que muestra cómo aislar una aorta de una rata, calcio etiqueta o NO en las células endoteliales o de músculo liso, y la imagen de los transitorios de calcio (o la producción de NO) utilizando un microscopio de dos fotones siguiente fisiológica o estímulos farmacológicos. Los beneficios de usar el método son multi-pliegue: 1) es posible simultáneaLy medir los transitorios de calcio en ambos células endoteliales y células del músculo liso en respuesta a diferentes estímulos; 2) le permite a uno a las células imagen endoteliales y células musculares lisas en su entorno nativo; 3) este método es muy sensible al calcio intracelular o ningún cambio y genera imágenes de alta resolución para mediciones precisas; y 4) enfoque descrito se puede aplicar a la medición de otras moléculas, tales como las especies reactivas del oxígeno. En resumen, la aplicación de dos fotones microscopía de emisión láser para controlar los transitorios de calcio y la producción de NO en las células endoteliales y de músculo liso de un vaso sanguíneo aislado haya facilitado datos cuantitativos de alta calidad y promovido nuestra comprensión de los mecanismos que regulan la función vascular.

Con el fin de evaluar con precisión la contribución de calcio a la fisiología vascular (vasodilatación y vasoconstricción), un protocolo fue diseñado para cargar colorantes de calcio en ambas células endoteliales y células del músculo liso en aortas intacto aislado. La experimental general que establezcan representado en la Figura 1, muestra la estrategia básica para el aislamiento y la preparación del recipiente antes de formación de imágenes. Brevemente, después del aislamiento de la aor...

Watch this Scientific Journal Video about Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta at JoVE.com

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

De dos fotones de imágenes de Ca intracelular<sup&gt; 2 +</sup&gt; Manejo y producción de óxido nítrico en el endotelio y las células del músculo liso de la aorta de rata aislada

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

two-photon-imaging-intracellular-ca2-handling-nitric-oxide-production

Research

JoVE Journal

Biology

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

11.2K vistas.  Medical College of Wisconsin. Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta. 

Video: Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

De dos fotones axotomía y de lapso de tiempo de imagen confocal en embriones de pez cebra en vivo

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Investigación

Biología

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Focal Ca 2 + Detección de transitorios en el músculo liso

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

El aislamiento de los Derechos Humanos de la arteria umbilical células musculares lisas (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

La medición de la función del músculo liso en el tejido aislado Bath-aplicaciones en Farmacología Investigación

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagen Ca Local<sup&gt; 2 +</sup&gt; Las señales en las células de mamífero en cultivo

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

En Detección de la cara del óxido nítrico y superóxido en la capa endotelial de las arterias intactas

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

De imagen confocal de medición de las fluctuaciones de calcio en el retículo sarcoplásmico de células musculares lisas cultivadas Uso de FRET a base de

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Aislamiento de murino coronaria células musculares lisas vasculares

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Desarrollo y caracterización de<em&gt; In Vitro</em&gt; Red de microvasos y mediciones cuantitativas de endotelial [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; Y la producción de óxido nítrico

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Lesión por aplastamiento aórtico murino: una eficacia<em&gt; In Vivo</em&gt; Modelo de proliferación de células musculares lisas y función endotelial

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...